protein structure determination - ut southwestern medical ......protein structure determination an...
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Protein Structure Determination
An Introduction
Chad A. Brautigam, Ph.D.Structural Biology Laboratory
UTSW Medical Center at Dallas
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What is “Protein Structure?”
…Arg-Lys-Ala-Gln-Trp-Cys-His-Ala-Asp…
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What is “Protein Structure?”
• It is the description of the three-dimensional architecture of a polypeptide.
• Our aim: to know accurately and precisely the position of every atom in a protein. That’s usually between 2000 and 10,000 atoms!
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Wherefore Art Thou, Protein Structure?
• To know a protein’s shape is to get a glimpse of its function.
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Sometimes, Structure = Function
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Other Times, Structure Suggests Function
A structure can give us some wonderful things,i.e. testable hypotheses!
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How Do We Obtain Protein Structures?
• Nuclear Magnetic Resonance (similar to MRI)
• Electron Crystallography• Neutron Crystallography• X-ray Crystallography
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In Order to Do Protein Crystallography, We
Need…
Protein Crystals!
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Protein Crystals
In order to obtain crystals of a protein, it is almostalways necessary to have pure protein.
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Protein Sources
• Animal, plant, or microbial tissues or cultures.
• Companies.• Overexpression.
–E. coli.– Insect cells.–Mammalian cell culture.
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Protein Purification
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Protein Purification
• We use the physical properties of the protein of interest to separate it from all contaminants.
• Chief among these properties are:–Charge.–Size.
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Quality Assessment
PAGE
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Quality AssessmentMass Spectrometry
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Now That We Have Pure Protein…
How do we crystallize it?
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Protein Crystallization
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Some Common Water-Withdrawing Chemicals
(Precipitants)• Polyethylene Glycol.• Ammonium Sulfate.• Sodium Chloride.• 1,4 Methyl Pentane Diol.• Ethyl Alcohol.
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Enough Theory-How Do We Actually Do
This?
Vapor Diffusion!
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Vapor Diffusion
“reservoir”
“Cover slip”“Drop”Crystal
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In Most Cases, Protein Crystals Are Not an End
They are the means by which we can determine a
protein’s structure.
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X-ray Diffraction by Crystals
X-rayGenerator X-ray
Detector
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An X-Ray Diffraction Pattern
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Electron Density
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Analysis of the Protein Structure
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Analysis of the Protein Structure
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Analysis of the Protein Structure